The present invention relates to a transcription factor found in filamentous fungi, especially in Aspergillii, DNA sequences coding for said factor, its transformation into and expression in fungal host organisms, and the use of said factor in such hosts for increasing the expression of a polypeptide of interest being produced by said host.
Transcription factors are well known proteins involved in the initiation of transcription. They have been studied intensively in many different organisms and have also been described in fungi. Dhawale and Lane (NAR (1993) 21 5537-5546) have recently compiled the transcription factors from fungi, including the filamentous fungi.
Many of the transcription factors are regulatory proteins; they bind to the promoter DNA and either activate or repress transcription as a response to stimuli to the cell.
The expression of the xcex1-amylase gene in A. oryzae is regulated in response to the available carbon source. The gene is expressed at its maximum when the organism is grown on starch or maltose (Lachmund et al. (1993) Current Microbiology 26 47-51; Tada et al. (1991) Mol. Gen. Genet 229 301-306). The expression of xcex1-amylase is regulated at the transcriptional level as shown by Lachmund et aL (supra), which strongly suggests that transcription factors are involved in the regulation, but so far no gene for such a factor has been identified.
The promoter of the xcex1-amylase gene has been studied by deletion analysis (Tada et al. (1991) Agric. Biol. Chem. 55 1939-1941; Tsuchiya et al. (1992) Biosci. Biotech. Biochem. 56 1849-1853; Nagata et al. (1993) Mol. Gen. Genet 237 251-260). The authors of these papers propose that a specific sequence of the promoter is responsible for the maltose induction. Nagata et al. (supra) used this sequence as a probe in a gel shift experiment to see whether any proteins from A. nidulans nuclear extracts were able to bind to the promoter sequence. Three such proteins were found, but no involvement of these proteins in expression was shown. None of the proteins have been purified or identified by other means. Their genes likewise remain unknown.
The present invention relates to a transcription factor regulating the expression of the xcex1-amylase promoter in filamentous fungi.
Accordingly, in a first aspect the invention relates to a DNA construct comprising a DNA sequence encoding a transcription factor of the invention, which DNA sequence comprises:
a) the transcription factor encoding part of the DNA sequence cloned into plasmid pToC320 present in E. coli ToC1058, DSM 10666, or
b) an analogue of the DNA sequence defined in a), which
i) is at least 60% homologous with the DNA sequence defined in a), or
ii) hybridizes with the same nucleotide probe as the DNA sequence defined in a), or
iii) encodes a transcription factor which is at least 50% homologous with the transcription factor encoded by a DNA sequence comprising the DNA sequence defined in a), or
iv) encodes a transcription factor which is immunologically reactive with an antibody raised against the purified transcription factor encoded by the DNA sequence defined in a), or
v) complements the mutation in ToC879, i.e. enables ToC879 to grow on cyclodextrin and produce lipase when transformed with said DNA sequence.
The full length genomic DNA sequence encoding a transcription factor has been derived from a strain of the filamentous fungus Aspergillus oryzae and has been cloned into plasmid pToC320 present in E. coli ToC1058, DSM 10666.
Said transcription factor encoding DNA sequence harboured in pToC320, DSM 10666, is believed to have the same sequence as that presented in SEQ ID NO: 1 and SEQ ID NO: 2. Accordingly, whenever reference is made to the transcription factor encoding part of the DNA sequence cloned into plasmid pToC320 present in DSM 10666 such reference is also intended to include the transcription factor encoding part of the DNA sequence presented in SEQ ID NO: 1 and SEQ ID NO: 2.
Accordingly, the terms xe2x80x9cthe transcription factor encoding part of the DNA sequence cloned into plasmid pToC320 present in DSM 10666xe2x80x9d and xe2x80x9cthe transcription factor encoding part of the DNA sequence presented in SEQ ID NO: 1 and SEQ ID NO: 2xe2x80x9d may be used interchangeably.
In further aspects the invention provides an expression vector harbouring the DNA construct of the invention, a cell comprising said DNA construct or said expression vector and a method of producing a peptide exhibiting transcription factor activity, which method comprises culturing said cell under conditions permitting the production of the transcription factor.
Such a transcription factor of the invention will typically originate from a filamentous fungus.
The term xe2x80x9cfilamentous fungusxe2x80x9d is intended to include the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and fungi imperfecti, including Hyphomycetes such as the genera Aspergillus, Penicillium, Trichoderma, Fusarium and Humicola.
The invention also relates to a method of producing a filamentous fungal host cell comprising the introduction of a DNA fragment coding for any such factor into a filamentous fungus wherein an xcex1-amylase promoter or a co-regulated promoter regulates the expression of a polypeptide of interest in a manner whereby said factor will be expressed in said fungus.
In a further aspect the invention relates to a method of producing a polypeptide of interest, the expression of which is regulated by an xcex1-amylase promoter or a co-regulated promoter, comprising growing a filamentous fungal host cell as described above under conditions conducive to the production of said factor and said polypeptide of interest, and recovering said polypeptide of interest.
Finally the invention relates to the use of said factor for regulating the expression of a polypeptide of interest in a filamentous fungus.
In this context, regulation means to change the conditions under which the factor of the invention is active. This could mean different pH, substrate, etc. regimes, whereby the resulting effect is an improved regulation of the expression of the protein of interest.
Furthermore, regulation also comprises events occurring in the growth phase of the fungus during which the transcription factor is active. Depending on the circumstances, both advancing and/or postponing the phase wherein the factor is active may enhance the expression and thus the yield.
In addition, using standard procedures known in the art, the specific DNA sequences involved in the binding of a transcription factor may be identified, thereby making it possible to insert such sequences into other promoters not normally regulated by the factor and enabling those promoters to be under the regulation of said factor.